1. Field of the Invention
The present invention is related to a monoclonal anti-idiotypic antibody directed against a Factor VIII inhibitory antibody which binds to the C1 domain of Factor VIII, as well as to a cell line producing this monoclonal anti-idiotypic antibody, to the use of this monoclonal anti-idiotypic antibody as medicament, and more particularly, to the use thereof for manufacturing a medicament for the treatment of haemophilia A.
2. Brief Description of the Related Art
Haemophilia A is a hereditary disease linked to an anomaly of chromosome X, which displays itself in the affected person by an inability to coagulate. This disease is the result of mutations on the gene of a protein involved in coagulation, the Factor VIII (FVIII) protein, which determine either a total absence of Factor VIII in the blood, or a partial deficit thereof.
Haemophilia A is the most common of insufficiencies affecting blood coagulation: in France 1 male in 5000 is affected, which represents 80% of patients suffering from haemophilia. The other type of haemophilia, haemophilia B, affects 20% of patients suffering from haemophilia; it is caused by a deficiency in an other clotting factor, known as Factor IX.
Present treatment of haemophilia (type A or B) consists of intravenous administration of the deficient or absent clotting factor. In France, Factor VIII for the treatment of haemophiliacs is available in form of blood derived medicaments provided by the Laboratoire Français du Fractionnement et des Biotechnologies (LFB) or by international pharmaceutical laboratories, as well as in form of recombinant medicaments prepared by genetic engineering methods. Effectively, the DNA coding Factor VIII has been isolated and expressed in mammalian cells (Wood et al., Nature (1984) 312: 330-337), and its amino acid sequence was deduced from cDNA.
Secreted Factor VIII (FVIII) is a glycoprotein with a molecular weight of 300 Kda (2332 amino acids), and plays a key role in the activation of intrinsic coagulation pathway. Inactive FVIII consists of six regions: A1 (residues 1-372), A2 (residues 373-740), B (residues 741-1648), A3 (residues 1690-2019), C1 (residues 2020-2172) and C2 (residues 2173-2332), from the N-terminal extremity to the C-terminal extremity. After being secreted, FVIII interacts with the von Willebrand Factor (vWF), which protects the FVIII against plasma proteases. FVIII dissociates from vWF upon cleavage by thrombin. This cleavage results in the elimination of the B domain and the formation of a heterodimer. FVIII circulates in plasma in this form. This heterodimer consists of a heavy chain (A1, A2) and of a light chain (A3, C1, C2).
When FVIII is infused to a haemophiliac patient, it binds to the von Willebrand Factor in the blood circulation of the patient. Activated Factor VIII acts as a co-factor of activated Factor IX, accelerating the conversion of Factor X into activated Factor X. Activated Factor X converts prothrombin into thrombin. Then the thrombin converts fibrinogen into fibrin, and clotting occurs.
The major problem encountered with Factor VIII administration is the appearance of antibodies directed against Factor VIII in the patient, referred to as <<inhibiting antibodies>>. These antibodies neutralize the procoagulant activity of Factor VIII, which is inactivated as soon as infused. Thus, the administered clotting factor is destroyed before bleeding can be stopped, which leads to a serious complication thus causing the treatment to be ineffective. Further, some genetically non-haemophiliac patients may develop inhibitors against endogenous Factor VIII: this is called acquired haemophilia.
Studies have shown that the anti-Factor VIII immune response is of the polyclonal IgG type belonging mostly to the IgG4 and IgG1 sub-class, and more rarely to IgG2. The IgG3 subclass is never represented. The light chain is often of Kappa type. The overrepresentation of IgG4 is more pronounced with haemophiliacs having a long-term established inhibitor. The C2 and A2 domains of the FVIII molecule are the favoured targets of the immune response although, in some cases, antibodies directed against the A3 domain are detected. When plasma of haemophiliac patients is passed through an immunoadsorption column with immobilized FVIII, it is possible to purify total anti-FVIII antibodies. The recovered amounts are often higher than 100 μg per 10 mg of total IgGs (Gilles J G et al. (1993) Blood; 82: 2452-2461). An animal model has been developed to study the formation of inhibitors of Factor VIII; rats immunized with human recombinant Factor VIII show a rapid immune response of the polyclonal type (Jarvis et al., Thromb Haemost. 1996 February; 75(2):318-25). The mechanisms by which anti-Factor VIII antibodies interfere with function of Factor VIII are numerous, and include interference with the proteolytic cleavage of Factor VIII and with the interaction of Factor VIII with different partners, such as von Willebrand Factor (vWF), phospholipids (PL), Factor IX, activated Factor X (FXa) or APC (Activated Protein C).
Several treatments allowing attenuation of the consequences of this immune response are available, such as for example treatments involving desmopressin, which is a synthetic hormone stimulating the production of Factor VIII, coagulation promoting agents, such as concentrates of prothrombin complexes or concentrates of activated prothrombin complexes, recombinant Factor VIIa, plasmapheresis and infusions of large or intermediary amounts of Factor VIII. Nevertheless, these methods are very expensive and of low efficacy.
Because of the complexity of the in vivo analysis of this immune polyclonal response, monoclonal antibodies directed against certain domains of Factor VIII have been isolated by some research teams. Thus, a human monoclonal antibody of the IgG4kappa type, LE2E9, has been isolated. This antibody is directed against the C1 domain of Factor VIII and inhibits the cofactor activity of Factor VIII and its binding to the von Willebrand Factor (Jacquemin et al., (2000) Blood 95:156-163). In the same way, a human monoclonal antibody directed against the C2 domain of Factor VIII, referred to as BO2C11 (IgG4kappa), produced from a library of memory B cells of a patient suffering from haemophilia A with inhibitors, has been isolated (Jacquemin et al., Blood 1998 Jul. 15; 92 (2):496-506). BO2C11 recognizes the C2 domain of Factor VIII, and inhibits its binding to von Willebrand Factor and to phospholipids. It completely inhibits the procoagulation activity of native and activated Factor VIII. A further example of monoclonal antibody is the BOIIB2 antibody directed against the A2 domain of Factor VIII. The BOIIB2 antibody inhibits 99% of Factor VIII activity. By binding to the A2 domain, it can interfere with and inhibit the binding of FIXa, which contains a low affinity binding site within this region of FVIII, and thus inhibits the enzyme activity of FIXa. The second conceivable way of action is its interference with the equilibrium between the heterodimeric form (A2:A1 and A3:C1:C2) of FVIII and the heterotrimeric form (A2 and A1 and A3:C1:C2) of FVIII by accelerating the dissociation of the A2 domain of these complexes, rendering them non-functional. (Ananyeva N M et al., (2004) Blood Coagul Fibrinolysis. Mar. 15(2):109-24. Review).
With the aid of these new tools, a further, more recent strategic struggle against the Factor VIII inhibitor antibodies has considered administering anti-idiotypic antibodies (antibodies having the ability to interact with the variable region of other antibodies) neutralizing the inhibitor antibodies (Saint-Rémy J M et al., (1999) Vox Sang; 77 (suppl 1): 21-24). A mouse anti-idiotypic antibody, known as 14C12, disclosed in the document WO 2004/014955, neutralizes in vivo, in a dose-dependent manner, the inhibitory properties of the anti-Factor VIII target antibody (monoclonal antibody BO2C11), which is directed against the C2 domain of Factor VIII. The anti-Factor VIII immune response being polyclonal, mouse anti-idiotypic antibodies directed against the A2 domain of Factor VIII have also been developed (and described in the patent application FR 05 08320). The A2 domain is a domain of 43 kD, the function of which is not well known but it has been demonstrated that inhibitory antibodies directed against the A2 domain of Factor VIII inhibit the function of Factor VIIIa by inhibiting the conversion of the complex FXase/FX in the transition state (Lollar et al., J Clin Invest. 1994 June; 93(6):2497-504, Fay et al., J Biol. Chem. 1996; 271(11): 6027-6032).
However, the immune response directed against Factor VIII is polyclonal, and, therefore, implies that inhibitory antibodies are directed against domains different from the A2 and C2 domains. Indeed, even if the study of epitopic specificities of anti-Factor VIII antibodies has shown that the majority of the inhibitors recognize limited zones of the Factor VIII molecule, located on the A2 domain of the heavy chain and/or on the C2 domain of the light chain, other epitopes are sometimes recognized. Indeed, some plasmas from patients contain antibodies capable to bind to the C1 domain of the light chain of Factor VIII (Moreau et al., 2000; 95(11):3435-441; Jacquemin et al., 2000; 95(1):156-162).
Thus, there is always a need for further tools enabling neutralization of other Factor VIII inhibitory antibodies directed against other domains of Factor VIII, in order to more completely neutralize the anti-Factor VIII polyclonal responses of haemophiliac patients.
Thus, the Applicant has attempted to develop a novel tool for treating haemophilia A enabling neutralization of inhibitory antibodies directed against the C1 domain of Factor VIII.